1H nuclear magnetic resonance study on equilibrium between two four-stranded solution conformations of short d(CnT).

Abstract

NMR analysis of d(C4T) showed the slow exchange between two distinct tetramers (each fully symmetric) in solution. For one tetramer, NOE cross-peak patterns characteristic of an i-motif structure (H1'-H1' and H6-H1'/H1'-H6) were observed between C1 and T5, indicating that this tetramer takes a completely intercalated conformation where the T5 residue is stacked on the C1.C1(+) pair of the other duplex (S-form). The other was found to be a tetramer in which one of the duplexes is shifted by one nucleotide unit (R-form), resulting in nonstacking 3' end thymidine residues and an equal number of stacked C.C+ pairs to that of the S-form. The same spectral features were observed for d(C3T) but neither for d(TC3) nor d(TC4), indicative of the critical role of the position of the thymidine residue in the tetrad isomerization. From NMR denaturation profiles, the S-forms were found to be more stable than the R-forms, and the linear relationship between the logarithm of the equilibrium constant (K = [tetramer]/[single]4) and the inverse of temperature (1/T) was confirmed for both forms, indicating conformity to the two-state transition model. Both enthalpy and entropy values of the formation of the S-form from four single strands were more negative than those of the R-form. The enthalpy term should contribute to the stabilization of the S-forms at low temperatures. The difference of the free energy values [DeltaG degrees(S-form) - DeltaG degrees(R-form)] was found to be -2.1 and -2.7 kJ.mol-1 at 20 degreesC for d(C4T) and d(C3T), respectively, explaining the higher stability of the S-forms. With increasing temperature, these two topologies were found to comparably exist at equilibrium in solution with slow exchange via dissociation to the single strands. A biological role of this topological isomerization is also suggested.